PROJECT SUMMARY Dysregulated homeostatic mechanisms that lead to an increased resistance to aqueous humor (AH) outflow through the trabecular pathway constitute the main cause for ocular hypertension (OHT), a key risk factor for an irreversible blinding disease, glaucoma. The extracellular matrix (ECM) is well-recognized to play a crucial role in modulation of AH outflow and intraocular pressure (IOP) via regulating the contractile, adhesive, fibrogenic and permeability characteristics of the trabecular outflow pathway cells. There still exists a fundamental gap in our understanding however, of how post-translational modifications of ECM proteins and ECM degrading enzymes might influence trabecular meshwork (TM) cell behavior, AH outflow and IOP. The broad objective of this proposal is to examine protein phosphorylation of ECM components and matrix metalloproteinases (MMPs), and determine how this modification modulates AH outflow and IOP, with the goal of uncovering novel insights into OHT etiology and developing efficacious strategies for glaucoma treatment. The scientific premise is that protein tyrosine kinases and phosphatases in the AH secreted by TM and other cell types play a vital role in homeostasis of AH outflow and IOP by modifying protein tyr-phosphorylation status of ECM and MMPs that in turn influences TM cell contractile, adhesive, fibrogenic and barrier activities. We hypothesize that deregulation of tyr-phosphorylation of ECM proteins and MMPs resulting from alterations in the levels of secretory tyrosine kinases and phosphatases may be a key etiological contribution to impaired AH outflow and elevated IOP in glaucoma. In support of this novel and paradigm shifting hypothesis, our recent studies detected vertebrate lonesome kinase (VLK) and phosphatase and tensin homolog (PTEN) proteins in human AH samples, and demonstrated that HTM cells secrete both enzymes. Significantly, VLK was shown to regulate ECM tyr-phosphorylation, and contractile and adhesive activities of TM cells, and induced by TGF-?2 and dexamethasone in human TM cells, both of which are known to induce OHT. Based on these promising and novel preliminary results, we have outlined three specific aims in this competing proposal, focused on mechanistic investigations into the role(s) of VLK in: 1) regulation of intracellular signaling pathways controlling TM cell contractile, cell-ECM & cell-cell adhesive, fibrogenic and permeability characteristics, 2) Tyr-phosphorylation of TM cell ECM and MMPs, ECM organization and TM cell stiffness, and 3) AH outflow and IOP, using human TM cells, human donor eyes and rats. These studies will utilize gene targeting, proteomics, biophysical, histological and physiological approaches, and include an assessment of changes in levels of VLK and PTEN in AH samples from glaucoma versus non-glaucoma (cataract) patients. Completion of the novel studies proposed in this application is expected to uncover new understanding on the role of tyr-phosphorylation of ECM proteins and MMPs in AH outflow and OHT etiology via altered outside-in signaling, and to identify new and efficacious treatment strategies for lowering IOP in glaucoma patients.